Aspartate kinase

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding an aspartate kinase. The invention also relates to the construction of a chimeric gene encoding all or a portion of the aspartate kinase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the aspartate kinase in a transformed host cell.

This application claims the benefit of U.S. Provisional Application No.60/172,944, filed Dec. 21, 1999.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingaspartate kinase in plants and seeds.

BACKGROUND OF THE INVENTION

Many vertebrates, including man, lack the ability to manufacture anumber of amino acids and therefore require these amino acids preformedin the diet. These are called essential amino acids. Human food andanimal feed, derived from many grains, are deficient in essential aminoacids, such as lysine, the sulfur amino acids methionine and cysteine,threonine and tryptophan. For example, in corn (Zea mays L.) lysine isthe most limiting amino acid for the dietary requirements of manyanimals. Soybean (Glycine max L.) meal is used as an additive tocorn-based animal feeds primarily as a lysine supplement. Thus, anincrease in the lysine content of either corn or soybean would reduce oreliminate the need to supplement mixed grain feeds with lysine producedvia fermentation of microbes. Furthermore, in corn the sulfur aminoacids are the third most limiting amino acids, after lysine andtryptophan, for the dietary requirements of many animals. The use ofsoybean meal, which is rich in lysine and tryptophan, to supplement cornin animal feed is limited by the low sulfur amino acid content of thelegume. Thus, an increase in the sulfur amino acid content of eithercorn or soybean would improve the nutritional quality of the mixturesand reduce the need for further supplementation through addition of moreexpensive methionine.

Efforts to improve the sulfur amino acid content of crops through plantbreeding have met with limited success on the laboratory scale and nosuccess on the commercial scale. A mutant corn line which had anelevated whole-kernel methionine concentration was isolated from corncells grown in culture by selecting for growth in the presence ofinhibitory concentrations of lysine plus threonine [Phillips et al.,Cereal Chem., (1985), 62, 213-218]. However, agronomically-acceptablecultivars have not yet been derived from this line and commercialized.Soybean cell lines with increased intracellular concentrations ofmethionine were isolated by selection for growth in the presence ofethionine [Madison and Thompson, Plant Cell Reports, (1988), 7,472-476], but plants were not regenerated from these lines.

Lysine, threonine, methionine, cysteine and isoleucine are amino acidsderived from aspartate. One approach to increasing the nutritionalquality of human foods and animal feed is to increase the production andaccumulation of specific free amino acids via genetic engineering of thebiosynthetic pathway that leads from aspartate to lysine, threonine,methionine, cysteine and isoleucine. However, few of the genes encodingenzymes that regulate this pathway in plants, especially corn, soybeansand wheat, are available. Alteration of the activity of enzymes in thispathway could lead to altered levels of lysine, threonine, methionine,cysteine and isoleucine. For instance, recombinant DNA and gene transfertechnologies have been applied to alter enzyme activity at key steps inthe amino acid biosynthetic pathway. The introduction into plants of afeedback-regulation-insensitive dihydrodipicolinic acid synthase(“DHDPS”) gene, which encodes an enzyme that catalyzes the firstreaction unique to the lysine biosynthetic pathway, has resulted in anincrease in the levels of free lysine in the leaves and seeds of thoseplants (Falco, U.S. Pat. No. 5,773,691; Glassman, U.S. Pat. No.5,258,300). Also, expression in plants of a bacterial lysC gene withaspartate kinase activity has resulted in an increase in threoninecontent of the seed (Karchi, et al. The Plant J. 3:721-727 (1993);Galili, et al., European Patent Application No. 0485970). However,expression of the lysC gene results in only a 6-7% increase in the levelof total threonine or methionine in the seed; thus, feed containing lysCtransgenic seeds still requires amino acid supplementation.

The organization of the pathway leading to biosynthesis of lysine,threonine, methionine, cysteine and isoleucine indicates thatover-expression or reduction of expression of several genes encoding keyregulatory enzymes of the pathway in corn, soybean, wheat and other cropplants could be used to alter levels of these amino acids in human foodand animal feed. For example, methionine, along with threonine, lysineand isoleucine, are amino acids derived from aspartate. The first stepin the pathway is the phosphorylation of aspartate by the enzymeaspartate kinase (Tang et al. (1997) Plant Mol Biol 34:287-293; Frankardet al. (1997) Plant Mol. Biol 34:233-242), and this enzyme has beenfound to be an important target for regulation of the pathway in manyorganisms. The aspartate family pathway is also believed to be regulatedat the branch-point reactions. For methionine the reduction of aspartylβ-semialdehyde by homoserine dehydrogenase (HDH) may be an importantpoint of control. Some aspartate kinases only carry aspartate kinaseactivity, in which case they are referred to as monofunctional, whereasthere are bifunctional proteins found in bacteria and plants that carryboth aspartate kinase and homoserine dehydrogenase enzymatic activitiesin two separate domains on one polypeptide. The first committed step tomethionine, the production of cystathionine from O-phosphohomoserine andcysteine by cystathionine γ-synthase (CS), appears to be an importantpoint of control of flux through the methionine pathway [Giovanelli etal., Plant Physiol., (1984), 77, 450-455]. The final step in methioninebiosynthesis is catalyzed by the enzyme5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase,also known as methionine synthase. Accordingly, availability of nucleicacid sequences encoding all or a portion of aspartate kinase wouldfacilitate development of nutritionally improved crop plants.

SUMMARY OF THE INVENTION

The present invention concerns an isolated polynucleotide comprising:(a) a first nucleotide sequence encoding a first polypeptide comprisingat least 50 or 100 amino acids, wherein the amino acid sequence of thefirst polypeptide and the amino acid sequence of SEQ ID NO:10 have atleast 95% identity based on the Clustal alignment method, (b) a secondnucleotide sequence encoding a second polypeptide comprising at least 95or 100 amino acids, wherein the amino acid sequence of the secondpolypeptide and the amino acid sequence of SEQ ID NO:2 have at least 90%or 95% identity based on the Clustal alignment method, (c) a thirdnucleotide sequence encoding a third polypeptide comprising at least 100amino acids, wherein the amino acid sequence of the third polypeptideand the amino acid sequence of SEQ ID NO:4 have at least 70%, 80%, 85%,90%, or 95% identity based on the Clustal alignment method, (d) a fourthnucleotide sequence encoding a fourth polypeptide comprising at least100 amino acids, wherein the amino acid sequence of the fourthpolypeptide and the amino acid sequence of SEQ ID NO:14 have at least80%, 85%, 90%, or 95% identity based on the Clustal alignment method,(e) a fifth nucleotide sequence encoding a fifth polypeptide comprisingat least 250 amino acids, wherein the amino acid sequence of the fifthpolypeptide and the amino acid sequence of SEQ ID NO: 12 have at least80%, 85%, 90%, or 95% identity based on the Clustal alignment method,(f) a sixth nucleotide sequence encoding a sixth polypeptide comprisingat least 400 amino acids, wherein the amino acid sequence of the sixthpolypeptide and the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8have at least 85%, 90%, or 95% identity based on the Clustal alignmentmethod, (g) a seventh nucleotide sequence encoding a seventh polypeptidecomprising at least 400 amino acids, wherein the amino acid sequence ofthe seventh polypeptide and the amino acid sequence of SEQ ID NO:16 haveat least 90% or 95% identity based on the Clustal alignment method, or(h) the complement of the first, second, third, fourth, fifth, sixth, orseventh nucleotide sequence, wherein the complement and the first,second, third, fourth, fifth, sixth, or seventh nucleotide sequencecontain the same number of nucleotides and are 100% complementary. Thefirst polypeptide preferably comprises the amino acid sequence of SEQ IDNO:10, the second polypeptide preferably comprises the amino acidsequence of SEQ ID NO:2, the third polypeptide preferably comprises theamino acid sequence of SEQ ID NO:4, the fourth polypeptide preferablycomprises the amino acid sequence of SEQ ID NO:14, the fifth polypeptidepreferably comprises the amino acid sequence of SEQ ID NO:12, the sixthpolypeptide preferably comprises the amino acid sequence of SEQ ID NO:6or SEQ ID NO:8, and the seventh polypeptide preferably comprises theamino acid sequence of SEQ ID NO:16. The first nucleotide sequencepreferably comprises the nucleotide sequence of SEQ ID NO:9, the secondnucleotide sequence preferably comprises the nucleotide sequence of SEQID NO: 1, the third nucleotide sequence preferably comprises thenucleotide sequence of SEQ ID NO:3, the fourth nucleotide sequencepreferably comprises the nucleotide sequence of SEQ ID NO:13, the fifthnucleotide sequence preferably comprises the nucleotide sequence of SEQID NO:11, the sixth nucleotide sequence preferably comprises thenucleotide sequence of SEQ ID NO:5 or SEQ ID NO:7, and the seventhnucleotide sequence preferably comprises the nucleotide sequence of SEQID NO:15. The first, second, third, fourth, fifth, sixth, and seventhpolypeptides preferably are aspartate kinases.

In a second embodiment, the present invention relates to a chimeric genecomprising any of the isolated polynucleotides of the present inventionoperably lined to a regulatory sequence, and a cell, a plant, and a seedcomprising the chimeric gene.

In a third embodiment, the present invention relates to a vectorcomprising any of the isolated polynucleotides of the present invention.

In a fourth embodiment, the present invention relates to an isolatedpolynucleotide fragment comprising a nucleotide sequence comprised byany of the polynucleotides of the present invention, wherein thenucleotide sequence contains at least 30, 40, or 60 nucleotides.

In a fifth embodiment, the present invention concerns an isolatedpolypeptide comprising: (a) a first amino acid sequence comprising atleast 50 or 100 amino acids, wherein the first amino acid sequence andthe amino acid sequence of SEQ ID NO: 10 have at least 95% identitybased on the Clustal alignment method, (b) a second amino acid sequencecomprising at least 95 or 100 amino acids, wherein the second amino acidsequence and the amino acid sequence of SEQ ID NO:2 have at least 90% or95% identity based on the Clustal alignment method, (c) a third aminoacid sequence comprising at least 100 amino acids, wherein the thirdamino acid sequence and the amino acid sequence of SEQ ID NO:4 have atleast 70%, 80%, 85%, 90%, or 95% identity based on the Clustal alignmentmethod, (d) a fourth amino acid sequence comprising at least 100 aminoacids, wherein the fourth amino acid sequence and the amino acidsequence of SEQ ID NO: 14 have at least 80%, 85%, 90%, or 95% identitybased on the Clustal alignment method, (e) a fifth amino acid sequencecomprising at least 250 amino acids, wherein the fifth amino acidsequence and the amino acid sequence of SEQ ID NO:12 have at least 80%,850%, 90%, or 95% identity based on the Clustal alignment method, (f) asixth amino acid sequence comprising at least 400 amino acids, whereinthe sixth amino acid sequence and the amino acid sequence of SEQ ID NO:6or SEQ ID NO:8 have at least 85%, 90%, or 95% identity based on theClustal alignment method, or (g) a seventh amino acid sequencecomprising at least 400 amino acids, wherein the seventh amino acidsequence and the amino acid sequence of SEQ ID NO:16 have at least 90%or 95% identity based on the Clustal alignment method. The first aminoacid sequence preferably comprises the amino acid sequence of SEQ IDNO:10, the second amino acid sequence preferably comprises the aminoacid sequence of SEQ ID NO:2, the third amino acid sequence preferablycomprises the amino acid sequence of SEQ ID NO:4, the fourth amino acidsequence preferably comprises the amino acid sequence of SEQ ID NO:14,the fifth amino acid sequence preferably comprises the amino acidsequence of SEQ ID NO:12, the sixth amino acid sequence preferablycomprises the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8, and theseventh amino acid sequence preferably comprises the amino acid sequenceof SEQ ID NO:16. The polypeptide preferably is an aspartate kinase.

In a sixth embodiment, the present invention relates to a method fortransforming a cell comprising transforming a cell with any of theisolated polynucleotides of the present invention, and the celltransformed by this method. Advantageously, the cell is eukaryotic,e.g., a yeast or plant cell, or prokaryotic, e.g., a bacterium.

In a seventh embodiment, the present invention relates to a method forproducing a transgenic plant comprising transforming a plant cell withany of the isolated polynucleotides of the present invention andregenerating a plant from the transformed plant cell, the transgenicplant produced by this method, and the seed obtained from thistransgenic plant.

In an eighth embodiment, the present invention relates to a virus,preferably a baculovirus, comprising any of the isolated polynucleotidesof the present invention or any of the chimeric genes of the presentinvention.

In a ninth embodiment, the invention relates to a method of selecting anisolated polynucleotide that affects the level of expression of anaspartate kinase polypeptide or enzyme activity in a host cell,preferably a plant cell, the method comprising the steps of: (a)constructing an isolated polynucleotide of the present invention or anisolated chimeric gene of the present invention; (b) introducing theisolated polynucleotide or the isolated chimeric gene into a host cell;(c) measuring the level of the aspartate kinase polypeptide or enzymeactivity in the host cell containing the isolated polynucleotide; and(d) comparing the level of the aspartate kinase polypeptide or enzymeactivity in the host cell containing the isolated polynucleotide withthe level of the aspartate kinase polypeptide or enzyme activity in thehost cell that does not contain the isolated polynucleotide.

In a tenth embodiment, the invention concerns a method of obtaining anucleic acid fragment encoding a substantial portion of an aspartatekinase polypeptide, preferably a plant aspartate kinase polypeptide,comprising the steps of: synthesizing an oligonucleotide primercomprising a nucleotide sequence of at least one of 60 preferably atleast one of 40, most preferably at least one of 30) contiguousnucleotides derived from a nucleotide sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, and 15, and thecomplement of such nucleotide sequences; and amplifying a nucleic acidfragment (preferably a cDNA inserted in a cloning vector) using theoligonucleotide primer. The amplified nucleic acid fragment preferablywill encode a substantial portion of an aspartate kinase polypeptideamino acid sequence.

In an eleventh embodiment, this invention relates to a method ofobtaining a nucleic acid fragment encoding all or a substantial portionof the amino acid sequence encoding an aspartate kinase polypeptidecomprising the steps of: probing a cDNA or genomic library with anisolated polynucleotide of the present invention; identifying a DNAclone that hybridizes with an isolated polynucleotide of the presentinvention; isolating the identified DNA clone; and sequencing the cDNAor genomic fragment that comprises the isolated DNA clone.

In a twelfth embodiment, this invention concerns a method for positiveselection of a transformed cell comprising: (a) transforming a host cellwith the chimeric gene of the present invention or an expressioncassette of the present invention; and (b) growing the transformed hostcell preferably a plant cell, such as a monocot or a dicot, underconditions which allow expression of the aspartate kinase polynucleotidein an amount sufficient to complement a null mutant to provide apositive selection means.

In a thirteenth embodiment, this invention relates to a method ofaltering the level of expression of an aspartate kinase in a host cellcomprising: (a) transforming a host cell with a chimeric gene of thepresent invention; and (b) growing the transformed host cell underconditions that are suitable for expression of the chimeric gene whereinexpression of the chimeric gene results in production of altered levelsof the aspartate kinase in the transformed host cell.

In a fourteenth embodiment, this invention relates to a method ofgenerating an aspartate kinase variant that has reduced sensitivity toinhibition by lysine and the variant produced by this method.

BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawing and Sequence Listing which forma part of this application.

FIG. 1 shows an alignment of the amino acid sequences of aspartatekinase encoded by nucleotide sequences derived from corn clonecho1c.pk002.k6 (SEQ ID NO:6), corn clone cpd1c.pk010.k1 (SEQ ID NO:8),and Glycine max (NCBI GenBank Identifier (GI) No. 5305740; SEQ IDNO:17). Amino acids which are conserved among all and at least twosequences with an amino acid at that position are indicated with anasterisk (*). Dashes are used by the program to maximize alignment ofthe sequences.

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. Table 1 also identifies the cDNA clonesas individual ESTs (“EST”), the sequences of the entire cDNA insertscomprising the indicated cDNA clones (“FIS”), contigs assembled from twoor more ESTs (“Contig”), contigs assembled from an FIS and one or moreESTs or PCR fragment sequence (“Contig*”), or sequences encoding theentire protein derived from an FIS, a contig, or an FIS and PCR fragmentsequence (“CGS”). Nucleotide SEQ ID NOs:3, 9, and 13 correspond tonucleotide SEQ ID NOs:1, 3, and 5, respectively, presented in U.S.Provisional Application No. 60/172,944, filed Dec. 21, 1999. Amino acidSEQ ID NOs:4, 10, and 14 correspond to amino acid SEQ ID NOs:2, 4, and6, respectively, presented in U.S. Provisional Application No.60/172,944, filed Dec. 21, 1999. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825. TABLE 1 Aspartate Kinase SEQ ID NO:Protein (Plant Source) Clone Designation Status (Nucleotide) (AminoAcid) Aspartate Kinase (Corn) bms1.pk0008.e5 FIS 1 2 Aspartate Kinase(Corn) cho1c.pk002.k6 EST 3 4 Aspartate Kinase (Corn) cho1c.pk002.k6(FIS) CGS 5 6 Aspartate Kinase (Corn) cpd1c.pk010.k1 (FIS) CGS 7 8Aspartate Kinase (Rice) rdr1f.pk005.f20 EST 9 10 Aspartate Kinase (Rice)rdr1f.pk005.f20 FIS 11 12 Aspartate Kinase (Wheat) wr1.pk0046.b11 EST 1314 Aspartate Kinase (Wheat) wr1.pk0046.b11 FIS 15 16

SEQ ID NO:17 sets forth the amino acid sequence of a precursormonofunctional aspartate kinase from Glycine max (NCBI GI No. 5305740).

SEQ ID NOS:18-21 are PCR primers used to amplify portions of the cDNAinsert in clone cpd1c.pk010.k1 to create an aspartate-kinase-encodingconstruct for expression in E. coli.

SEQ ID NOS:22 and 23 are PCR primers used to introduce a site-specificmutation to change S (serine) to L (leucine) in the corn mono functionalaspartate kinase as described in Example 8.

SEQ ID NO:24 is a PCR primer which was used with SEQ ID NO:21 togenerate a 380 bp PCR fragment which has Nco I sites on both ends andcontains the 5′ end of the coding sequence including the plantchloroplast targeting sequence.

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment”are used interchangeably herein. These terms encompass nucleotidesequences and the like. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least one of 60contiguous nucleotides, preferably at least one of 40 contiguousnucleotides, most preferably one of at least 30 contiguous nucleotidesderived from SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, or 15, or the complementof such sequences.

The term “isolated” polynucleotide refers to a polynucleotide that issubstantially free from other nucleic acid sequences, such as otherchromosomal and extrachromosomal DNA and RNA, that normally accompany orinteract with it as found in its naturally occurring environment.Isolated polynucleotides may be purified from a host cell in which theynaturally occur. Conventional nucleic acid purification methods known toskilled artisans may be used to obtain isolated polynucleotides. Theterm also embraces recombinant polynucleotides and chemicallysynthesized polynucleotides.

The term “recombinant” means, for example, that a nucleic acid sequenceis made by an artificial combination of two otherwise separated segmentsof sequence, e.g., by chemical synthesis or by the manipulation ofisolated nucleic acids by genetic engineering techniques.

As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof. Theterms “substantially similar” and “corresponding substantially” are usedinterchangeably herein.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least one of 30 contiguous nucleotides derived from theinstant nucleic acid fragment can be constructed and introduced into aplant or plant cell. The level of the polypeptide encoded by theunmodified nucleic acid fragment present in a plant or plant cellexposed to the substantially similar nucleic fragment can then becompared to the level of the polypeptide in a plant or plant cell thatis not exposed to the substantially similar nucleic acid fragment.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby using nucleic acid fragments that do not share 100% sequence identitywith the gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least one of 60 (preferably at least one of40, most preferably at least one of 30) contiguous nucleotides derivedfrom a nucleotide sequence selected from the group consisting of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, and 15, and the complement of such nucleotidesequences may be used in methods of selecting an isolated polynucleotidethat affects the expression of an aspartate kinase polypeptide in a hostcell. A method of selecting an isolated polynucleotide that affects thelevel of expression of a polypeptide in a virus or in a host cell(eukaryotic, such as plant or yeast, prokaryotic such as bacterial) maycomprise the steps of: constructing an isolated polynucleotide of thepresent invention or an isolated chimeric gene of the present invention;introducing the isolated polynucleotide or the isolated chimeric geneinto a host cell; measuring the level of a polypeptide or enzymeactivity in the host cell containing the isolated polynucleotide; andcomparing the level of a polypeptide or enzyme activity in the host cellcontaining the isolated polynucleotide with the level of a polypeptideor enzyme activity in a host cell that does not contain the isolatedpolynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min. then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30min. A more preferred set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are at least about 70%identical, preferably at least about 80% identical to the amino acidsequences reported herein. Preferred nucleic acid fragments encode aminoacid sequences that are at least about 85% identical to the amino acidsequences reported herein. More preferred nucleic acid fragments encodeamino acid sequences that are at least about 90% identical to the aminoacid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are at least about 95%identical to the amino acid sequences reported herein. Suitable nucleicacid fragments not only have the above identities but typically encode apolypeptide having at least 50 amino acids, preferably at least 95 or atleast 100 amino acids, more preferably at least 150 amino acids, stillmore preferably at least 200 amino acids, and most preferably at least250 or at least 400 amino acids. Sequence alignments and percentidentity calculations were performed using the Megalign program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method were KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of the nucleotide sequence to reflectthe codon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign-gene” refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or may be composed ofdifferent elements derived from different promoters found in nature, ormay even comprise synthetic nucleotide segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmentalconditions. Promoters which cause a nucleic acid fragment to beexpressed in most cell types at most times are commonly referred to as“constitutive promoters”. New promoters of various types useful in plantcells are constantly being discovered; numerous examples may be found inthe compilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

“Translation leader sequence” refers to a nucleotide sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

“3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to DNA that is complementary toand derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase I. “Sense-RNA” refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single polynucleotide so that the functionof one is affected by the other. For example, a promoter is operablylinked with a coding sequence when it is capable of affecting theexpression of that coding sequence (i.e., that the coding sequence isunder the transcriptional control of the promoter). Coding sequences canbe operably linked to regulatory, sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

A “protein” or “polypeptide” is a chain of amino acids arranged in aspecific order determined by the coding sequence in a polynucleotideencoding the polypeptide. Each protein or polypeptide has a uniquefunction.

“Altered levels” or “altered expression” refers to the production ofgene product(s) in transgenic organisms in amounts or proportions thatdiffer from that of normal or non-transformed organisms.

“Null mutant” refers here to a host cell which either jacks theexpression of a certain polypeptide or expresses a polypeptide which isinactive or does not have any detectable expected enzymatic function.

“Mature protein” or the term “mature” when used in describing a proteinrefers to a post-translationally processed polypeptide; i.e., one fromwhich any pre- or propeptides present in the primary translation producthave been removed. “Precursor protein” or the term “precursor” when usedin describing a protein refers to the primary product of translation ofmRNA; i.e., with pre- and propeptides still present. Pre- andpropeptides may be but are not limited to intracellular localizationsignals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear location signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Thus, isolated polynucleotides of thepresent invention can be incorporated into recombinant constructs,typically DNA constructs, capable of introduction into and replicationin a host cell. Such a construct can be a vector that includes areplication system and sequences that are capable of transcription andtranslation of a polypeptide-encoding sequence in a given host cell. Anumber of vectors suitable for stable transfection of plant cells or forthe establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Flevin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

“PCR” or “polymerase chain reaction” is well known by those skilled inthe art as a technique used for the amplification of specific DNAsegments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

The present invention concerns an isolated polynucleotide comprising:(a) a nucleotide sequence encoding a first polypeptide comprising atleast 50 or 100 amino acids, wherein the amino acid sequence of thefirst polypeptide and the amino acid sequence of SEQ ID NO:10 have atleast 95% identity based on the Clustal alignment method, (b) a secondnucleotide sequence encoding a second polypeptide comprising at least 95or 100 amino acids, wherein the amino acid sequence of the secondpolypeptide and the amino acid sequence of SEQ ID NO:2 have at least 90%or 95% identity based on the Clustal alignment method, (c) a thirdnucleotide sequence encoding a third polypeptide comprising at least 100amino acids, wherein the amino acid sequence of the third polypeptideand the amino acid sequence of SEQ ID NO:4 have at least 70%, 80%, 85%,90%, or 95% identity based on the Clustal alignment method, (d) a fourthnucleotide sequence encoding a fourth polypeptide comprising at least100 amino acids, wherein the amino acid sequence of the fourthpolypeptide and the amino acid sequence of SEQ ID NO: 14 have at least80%, 85%, 90%, or 95% identity based on the Clustal alignment method,(e) a fifth nucleotide sequence encoding a fifth polypeptide comprisingat least 250 amino acids, wherein the amino acid sequence of the fifthpolypeptide and the amino acid sequence of SEQ ID NO:12 have at least80%, 85%, 90%, or 95% identity based on the Clustal alignment method,(f) a sixth nucleotide sequence encoding a sixth polypeptide comprisingat least 400 amino acids, wherein the amino acid sequence of the sixthpolypeptide and the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8have at least 85%, 90%, or 95% identity based on the Clustal alignmentmethod, (g) a seventh nucleotide sequence encoding a seventh polypeptidecomprising at least 400 amino acids, wherein the amino acid sequence ofthe seventh polypeptide and the amino acid sequence of SEQ ID NO:16 haveat least 90% or 95% identity based on the Clustal alignment method, or(h) the complement of the first, second, third, fourth, fifth, sixth, orseventh nucleotide sequence, wherein the complement and the first,second, third, fourth, fifth, sixth, or seventh nucleotide sequencecontain the same number of nucleotides and are 100% complementary. Thefirst polypeptide preferably comprises the amino acid sequence of SEQ IDNO:10, the second polypeptide preferably comprises the amino acidsequence of SEQ ID NO:2, the third polypeptide preferably comprises theamino acid sequence of SEQ ID NO:4, the fourth polypeptide preferablycomprises the amino acid sequence of SEQ ID NO:14, the fifth polypeptidepreferably comprises the amino acid sequence of SEQ ID NO:12, the sixthpolypeptide preferably comprises the amino acid sequence of SEQ ID NO:6or SEQ ID NO:8, and the seventh polypeptide preferably comprises theamino acid sequence of SEQ ID NO:16. The first nucleotide sequencepreferably comprises the nucleotide sequence of SEQ ID NO:9, the secondnucleotide sequence preferably comprises the nucleotide sequence of SEQID NO:1, the third nucleotide sequence preferably comprises thenucleotide sequence of SEQ ID NO:3, the fourth nucleotide sequencepreferably comprises the nucleotide sequence of SEQ ID NO:13, the fifthnucleotide sequence preferably comprises the nucleotide sequence of SEQID NO:11, the sixth nucleotide sequence preferably comprises thenucleotide sequence of SEQ ID NO:5 or SEQ ID NO:7, and the seventhnucleotide sequence preferably comprises the nucleotide sequence of SEQID NO:15. The first, second, third, fourth, fifth, sixth, and seventhpolypeptides preferably are aspartate kinases.

Nucleic acid fragments encoding at least a portion of several aspartatekinases have been isolated and identified by comparison of random plantcDNA sequences to public databases containing nucleotide and proteinsequences using the BLAST algorithms well known to those skilled in theart. The nucleic acid fragments of the instant invention may be used toisolate cDNAs and genes encoding homologous proteins from the same orother plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other aspartate kinases, either as cDNAs orgenomic DNAs, could be isolated directly by using all or a portion ofthe instant nucleic acid fragments as DNA hybridization probes to screenlibraries from any desired plant employing methodology well known tothose skilled in the art. Specific oligonucleotide probes based upon theinstant nucleic acid sequences can be designed and synthesized bymethods known in the art (Maniatis). Moreover, an entire sequence can beused directly to synthesize DNA probes by methods known to the skilledartisan such as random primer DNA labeling, nick translation,end-labeling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers can be designed andused to amplify a part or all of the instant sequences. The resultingamplification products can be labeled directly during amplificationreactions or labeled after amplification reactions, and used as probesto isolate full length cDNA or genomic fragments under conditions ofappropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).Consequently, a polynucleotide comprising a nucleotide sequence of atleast one of 60 (preferably one of at least 40, most preferably one ofat least 30) contiguous nucleotides derived from a nucleotide sequenceselected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13,and 15, and the complement of such nucleotide sequences may be used insuch methods to obtain a nucleic acid fragment encoding a substantialportion of an amino acid sequence of a polypeptide.

The present invention relates to a method of obtaining a nucleic acidfragment encoding a substantial portion of an aspartate kinasepolypeptide, preferably a substantial portion of a plant aspartatekinase polypeptide, comprising the steps of: synthesizing anoligonucleotide primer comprising a nucleotide sequence of at least oneof 60 (preferably at least one of 40, most preferably at least one of30) contiguous nucleotides derived from a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, and 15,and the complement of such nucleotide sequences; and amplifying anucleic acid fragment (preferably a cDNA inserted in a cloning vector)using the oligonucleotide primer. The amplified nucleic acid fragmentpreferably will encode a portion of an aspartate kinase polypeptide.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1-34; Maniatis).

In another embodiment, this invention concerns viruses and host cellscomprising either the chimeric genes of the invention as describedherein or an isolated polynucleotide of the invention as describedherein. Examples of host cells which can be used to practice theinvention include, but are not limited to, yeast, bacteria, and plants.

As was noted above, the nucleic acid fragments of the instant inventionmay be used to create transgenic plants in which the disclosedpolypeptides are present at higher or lower levels than normal or incell types or developmental stages in which they are not normally found.This would have the effect of altering the level of free amino acids(e.g., aspartate, threonine, lysine, and methionine) in those plants.Using these nucleic acid fragments that encode aspartate kinase,variants that have reduced sensitivity to lysine or another amino acid(e.g., threonine) may be generated by a variety of methods (e.g., themethod described in U.S. Pat. No. 5,773,691) such that the aspartatekinase may continue to be active in the presence of high levels oflysine or another amino acid (e.g., threonine), leading to theaccumulation of lysine and/or threonine in the seeds of transformedplants.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.The chimeric gene may comprise promoter sequences and translation leadersequences derived from the same genes. 3′Non-coding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene may also comprise one or more introns in order tofacilitate gene expression.

Plasmid vectors comprising the instant isolated polynucleotide (orchimeric gene) may be constructed. The choice of plasmid vector isdependent upon the method that will be used to transform host plants.The skilled artisan is well aware of the genetic elements that must bepresent on the plasmid vector in order to successfully transform, selectand propagate host cells containing the chimeric gene. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by directing the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) with or without removingtargeting sequences that are already present. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of use may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds. For example, one canscreen by looking for changes in gene expression by using antibodiesspecific for the protein encoded by the gene being suppressed, or onecould establish assays that specifically measure enzyme activity. Apreferred method will be one which allows large numbers of samples to beprocessed rapidly, since it will be expected that a large number oftransformants will be negative for the desired phenotype.

In another embodiment, the present invention concerns an isolatedpolypeptide comprising: (a) a first amino acid sequence comprising atleast 50 or 100 amino acids, wherein the first amino acid sequence andthe amino acid sequence of SEQ ID NO:10 have at least 95% identity basedon the Clustal alignment method, (b) a second amino acid sequencecomprising at least 95 or 100 amino acids, wherein the second amino acidsequence and the amino acid sequence of SEQ ID NO:2 have at least 90% or95% identity based on the Clustal alignment method, (c) a third aminoacid sequence comprising at least 100 amino acids, wherein the thirdamino acid sequence and the amino acid sequence of SEQ ID NO:4 have atleast 70%, 80%, 85%, 90%, or 95% identity based on the Clustal alignmentmethod, (d) a fourth amino acid sequence comprising at least 100 aminoacids, wherein the fourth amino acid sequence and the amino acidsequence of SEQ ID NO:14 have at least 80%, 85%, 90%, or 95% identitybased on the Clustal alignment method, (e) a fifth amino acid sequencecomprising at least 250 amino acids, wherein the fifth amino acidsequence and the amino acid sequence of SEQ ID NO:12 have at least 80%,85%, 90%, or 95% identity based on the Clustal alignment method, (f) asixth amino acid sequence comprising at least 400 amino acids, whereinthe sixth amino acid sequence and the amino acid sequence of SEQ ID NO:6or SEQ ID NO:8 have at least 85%, 90%, or 95% identity based on theClustal alignment method, or (g) a seventh amino acid sequencecomprising at least 400 amino acids, wherein the seventh amino acidsequence and the amino acid sequence of SEQ ID NO:16 have at least 90%or 95% identity based on the Clustal alignment method. The first aminoacid sequence preferably comprises the amino acid sequence of SEQ IDNO:10, the second amino acid sequence preferably comprises the aminoacid sequence of SEQ ID NO:2, the third amino acid sequence preferablycomprises the amino acid sequence of SEQ ID NO:4, the fourth amino acidsequence preferably comprises the amino acid sequence of SEQ ID NO:14,the fifth amino acid sequence preferably comprises the amino acidsequence of SEQ ID NO:12, the sixth amino acid sequence preferablycomprises the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8, and theseventh amino acid sequence preferably comprises the amino acid sequenceof SEQ ID NO:16. The polypeptide preferably is an aspartate kinase.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to these proteins by methods wellknown to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded aspartate kinase. An example of a vector for high levelexpression of the instant polypeptides in a bacterial host is provided(Example 6).

All or a substantial portion of the polynucleotides of the instantinvention may also be used as probes for genetically and physicallymapping the genes that they are a part of, and used as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics I:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Res.5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptide.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptide can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various corn (Zea mays), rice(Oryza sativa), and wheat (Triticum aestivum) tissues were prepared. Thecharacteristics of the libraries are described below. TABLE 2 cDNALibraries from Corn, Rice, and Wheat Library Tissue Clone bms1 Corn(BMS) Cell Culture 1 Day After bms1.pk0008.e5 Subculture cho1c CornEmbryo (Alexho Synthetic High cho1c.pk002.k6 Oil) 20 Days AfterPollination cpd1c Corn Pooled BMS Treated with Chemicals cpd1c.pk010.k1Related to Protein Kinases* rdr1f Developing Root of 10 Day Old Ricerdr1f.pk005.f20 Plant wr1 Root From 7 Day Old Light Grown wr1.pk0046.b11Wheat Seedling*Chemicals used included 1,2-didecanoyl rac glycerol, straurosporine,K-252a, A3, H-7, olomoucine, and rapamycin, all of which arecommercially available from Calbiochem-Novabiochem Corp.(1-800-628-8470)

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Full-insert sequence (FIS) data is generated utilizing a modifiedtransposition protocol. Clones identified for FIS are recovered fromarchived glycerol stocks as single colonies, and plasmid DNAs areisolated via alkaline lysis. Isolated DNA templates are reacted withvector primed M13 forward and reverse oligonucleotides in a PCR-basedsequencing reaction and loaded onto automated sequencers. Confirmationof clone identification is performed by sequence alignment to theoriginal EST sequence from which the FIS request is made.

Confirmed templates are transposed via the Primer Island transpositionkit (PE Applied Biosystems, Foster City, Calif.) which is based upon theSaccharomyces cerevisiae Ty1 transposable element (Devine and Boeke(1994) Nucleic Acids Res. 22:3765-3772). The in vitro transpositionsystem places unique binding sites randomly throughout a population oflarge DNA molecules. The transposed DNA is then used to transform DH10Belectro-competent cells (Gibco BRL/Life Technologies, Rockville, Md.)via electroporation. The transposable element contains an additionalselectable marker (named DHFR; Fling and Richards (1983) Nucleic AcidsRes. 11:5147-5158), allowing for dual selection on agar plates of onlythose subclones containing the integrated transposon. Multiple subclonesare randomly selected from each transposition reaction, plasmid DNAs areprepared via alkaline lysis, and templates are sequenced (ABI Prismdye-terminator ReadyReaction mix) outward from the transposition eventsite, utilizing unique primers specific to the binding sites within thetransposon.

Sequence data is collected (ABI Prism Collections) and assembled usingPhred/Phrap (P. Green, University of Washington, Seattle). Phrep/Phrapis a public domain software program which re-reads the ABI sequencedata, re-calls the bases, assigns quality values, and writes the basecalls and quality values into editable output files. The Phrap sequenceassembly program uses these quality values to increase the accuracy ofthe assembled sequence contigs. Assemblies are viewed by the Consedsequence editor (D. Gordon, University of Washington, Seattle).

Example 2 Identification of cDNA Clones

cDNA clones encoding aspartate kinase were identified by conductingBLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol.Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches forsimilarity to sequences contained in the BLAST “nr” database (comprisingall non-redundant GenBank CDS translations, sequences derived from the3-dimensional structure Brookhaven Protein Data Bank, the last majorrelease of the SWISS-PROT protein sequence database, EMBL, and DDBJdatabases). The cDNA sequences obtained in Example 1 were analyzed forsimilarity to all publicly available DNA sequences contained in the “nr”database using the BLASTN algorithm provided by the National Center forBiotechnology Information (NCBI). The DNA sequences were translated inall reading frames and compared for similarity to all publicly availableprotein sequences contained in the “nr” database using the BLASTXalgorithm (Gish and States (1993) Nat. Genet. 3:266-272) provided by theNCBI. For convenience, the P-value (probability) of observing a match ofa cDNA sequence to a sequence contained in the searched databases merelyby chance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

ESTs submitted for analysis are compared to the genbank database asdescribed above. ESTs that contain sequences more 5- or 3-prime can befound by using the BLASTn algorithm (Altschul et al (1997) Nucleic AcidsRes. 25:3389-3402.) against the DuPont proprietary database comparingnucleotide sequences that share common or overlapping regions ofsequence homology. Where common or overlapping sequences exist betweentwo or more nucleic acid fragments, the sequences can be assembled intoa single contiguous nucleotide sequence, thus extending the originalfragment in either the 5 or 3-prime direction. Once the most 5-prime ESTis identified, its complete sequence can be determined by Full InsertSequencing as described in Example 1. Homologous genes belonging todifferent species can be found by comparing the ammo acid sequence of aknown gene (from either a proprietary source or a public database)against an EST database using the tBLASTn algorithm. The tBLASTnalgorithm searches an amino acid query against a nucleotide databasethat is translated in all 6 reading frames. This search allows fordifferences in nucleotide codon usage between different species, and forcodon degeneracy.

Example 3 Characterization of cDNA Clones Encoding Aspartate Kinase

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs toaspartate kinase from Oryza sativa (NCBI GenBank Identifier (GI) No.7798569), Arabidopsis thaliana (NCBI GI Nos. 4376158 and 7529283), orGlycine max (NCBI GI No. 5305740). Shown in Table 3 are the BLASTresults for individual ESTs (“EST”), the sequences of the entire cDNAinserts comprising the indicated cDNA clones (“FIS”), the sequences ofcontigs assembled from two or more ESTs (“Contig”), sequences of contigsassembled from an FIS and one or more ESTs (“Contig*”), or sequencesencoding an entire protein derived from an FIS, a contig, or an FIS andPCR (“CGS”): TABLE 3 BLAST Results for Sequences Encoding PolypeptidesHomologous to Aspartate Kinase BLAST Results Clone Status NCBI GI No.pLog Score cho1c.pk002.k6 EST 4376158 19.30 rdr1f.pk005.f20 EST 530574054.70 wr1.pk0046.b11 EST 5305740 48.70

The sequence of the entire cDNA insert in the clones listed in Table 3was determined. Further sequencing and searching of the DuPontproprietary database allowed the identification of other corn clonesencoding aspartate kinase. The BLASTX search using the EST sequencesfrom clones listed in Table 4 revealed similarity of the polypeptidesencoded by the cDNAs to aspartate kinase from Oryza saliva (NCBI GI No.7798569), Arabidopsis thaliana (NCBI GI Nos. 4376158 and 7529283), orGlycine max (NCBI GI No. 5305740). Shown in Table 4 are the BLASTresults for individual ESTs (“EST”), the sequences of the entire cDNAinserts comprising the indicated cDNA clones (“FIS”), sequences ofcontigs assembled from two or more ESTs (“Contig”), sequences of contigsassembled from an FIS and one or more ESTs (“Contig*”), or sequencesencoding the entire protein derived from an FIS, a contig, or an FIS andPCR (“CGS”): TABLE 4 BLAST Results for Sequences Encoding PolypeptidesHomologous to Aspartate Kinase BLAST Results Clone Status NCBI GI No.pLog Score bms1.pk0008.e5 FIS 7798569 32.70 cho1c.pk002.k6 (FIS) CGS5305740 >180.00 cpd1c.pk010.k1 (FIS) CGS 5305740 >180.00 rdr1f.pk005.f20FIS 7529283 100.00 wr1.pk0046.b11 FIS 7529283 >180.00

FIG. 1 presents an alignment of the amino acid sequences set forth inSEQ ID NOs:6 and 8 and the Glycine max sequence (NCBI GI No. 5305740;SEQ ID NO:17). The data in Table 5 represents a calculation of thepercent identity of the amino acid sequences set forth in SEQ ID NOs:6and 8 and the Glycine max sequence (NCBI GI No. 5305740; SEQ ID NO:17).TABLE 5 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toAspartate Kinase Percent Identity to SEQ ID NO. NCBI GI No. 5305740; SEQID NO: 17 6 66.4 8 68.8

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of an aspartate kinase. These sequencesrepresent the first corn, rice and wheat sequences encoding aspartatekinase known to Applicant.

Example 4 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptide insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL1-Blue (EpicurianColi XL-1 Blue™; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (Sequenase™DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a chimeric gene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptide, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1:5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 5 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can be maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptide and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 6 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified 1% low melting agarose gel. Buffer and agarose contain 10 μg/mlethidium bromide for visualization of the DNA fragment. The fragment canthen be purified from the agarose gel by digestion with GELase™(Epicentre Technologies, Madison, Wis.) according to the manufacturer'sinstructions, ethanol precipitated, dried and resuspended in 20 μL ofwater. Appropriate oligonucleotide adapters may be ligated to thefragment using T4 DNA ligase (New England Biolabs (NEB); Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptide are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

Example 7 Functional Analysis of Aspartate Kinase Encoded by Clonecpd1c.pk010.k1

High level expression of the corn mono-functional aspartate kinase cDNAin clone cpd1c.pk010.k1 was accomplished in E. coli using thecommercially available expression vector pTrcHis from Invitrogen. Thecorn aspartate kinase cDNA in clone cpd1c.pk010.k1 was modified forinsertion into the expression vectors using PCR.

Cloning monofunctional corn aspartate kinase into expression vectorsrequired two steps. First, a portion of the corn mono-functionalaspartate kinase cDNA in clone cpd1c.pk010.k1 was amplified via PCRusing the following primers, to create a Kpn I site after the stopcodon: Oligo 1: 5′-CTCTCTGCCATGGGGAA-3′ (SEQ ID NO:18) Oligo 2:5′-GACTGGTACCTCAGCCCACGAGTAGGT-3′ (SEQ ID NO:19)

The resulting PCR fragment, designated PCR fragment 1, was digested withNco I and Kpn I and ligated into pTrcHis cut with the same enzymes. Thena different portion of the corn mono-functional aspartate kinase cDNA inclone cpd1c.pk010.k1 was amplified via PCR using the following primers,to remove the chloroplast transit sequence and create a NcoI-NcoIfragment: Oligo 9: 5′-GACTCCATGGAGGGATTGGGGGA-3′ (SEQ ID NO:20) Oligo 8:5′-GTTTTCCCCATGGCAGAGA-3′ (SEQ ID NO:21)The resulting PCR fragment, designated PCR fragment 3, was digested withNco I and ligated into the pTrcHis-based expression vector containing aportion of cpd1c.pk010.k1 cDNA described above that was also cut withNco I. Insertion of the Nco I fragment in the proper orientation wasdetermined by sequencing of the inserted DNA. The resulting plasmid withcDNA encoding full-length monofunctional corn aspartate kinase withoutchloroplast transit sequence in the pTrcHis vector was designatedpBT994.

To establish that the cloned monofunctional corn aspartate kinase cDNAwas functional, pBT994 was transformed into E. coli strain Gif106M1 (E.coli Genetic Stock Center strain CGSC-5074) which has mutations in eachof the three E. coli aspartate kinase genes [Theze et al. (1974) J.Bacteriol. 117:133-143]. Because this stain lacks all aspartate kinaseactivity, it requires lysine, threonine and methionine for growth M9media [see Sambrook et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press], supplemented with thearginine, isoleucine and valine, also required for Gif106M1 growth, wasused. In the pBT994 transformed strain the nutritional requirement forlysine, threonine and methionine was relieved demonstrating that thecloned monofunctional corn aspartate kinase cDNA encoded functionalaspartate kinase.

Example 8 Generation of Corn Aspartate Kinase with Reduced Sensitivityto Lysine

In order to use the monofunctional corn aspartate kinase to increase theproduction of the amino acid end-products of the pathway, i.e. lysine,threonine and methionine, it was desirable to create a mutant form ofthe enzyme that is insensitive to inhibition by lysine. Two approachesto accomplish this have been used.

One method to create a lysine-resistant mutant corn monofunctionalaspartate kinase relied on a procedure analogous to the procedure usedpreviously, and described in U.S. Pat. No. 5,773,691, to select mutantsin the E. coli lysC gene, which encodes E. coli monofunctional aspartatekinase.

Addition of lysine at a concentration of approximately 0.2 mM to thegrowth medium inhibits the growth of Gif106M1 transformed with pBT994.This inhibition is reversed by addition of threonine plus methionine tothe growth media. These results indicated that corn monofunctionalaspartate kinase could be inhibited by exogenously added lysine leadingto starvation for the other amino acids derived from aspartate. Thisproperty of pBT994 transformed Gif106M1 was used to select for mutationsthat encode lysine-insensitive monofunctional corn aspartate kinase.

Single colonies of Gif106M1 transformed with pBT994 were picked andresuspended in 200 μL of a mixture of 100 μL 1% lysine plus 100 μL of M9media. The entire cell suspension containing 10⁷-10⁸ cells was spread ona petri dish containing M9 media supplemented with the arginine,isoleucine, and valine. Sixteen petri dishes were thus prepared. From 1to 20 colonies appeared on 11 of the 16 petri dishes. One or twocolonies were picked and retested for lysine resistance and from thistest several independent lysine-resistant clones were obtained. PlasmidDNA was prepared from eight of these and re-transformed into Gif106M1 todetermine whether the lysine resistance determinant was plasmid-borne.Three of the eight plasmid DNAs yielded lysine-resistant coloniesindicating that they carry mutations in corn monofunctional AK that makethe enzyme less sensitive to lysine inhibition.

A second method used PCR mutagenesis to create a site-specific mutationin the corn monofunctional aspartate kinase gene that reduces theencoded enzyme's sensitivity to inhibition by L-lysine. The particularamino acid substitutions to yield lysine-insensitive monofunctional cornaspartate kinase were based upon the homology that was discoveredbetween monofunctional corn aspartate kinase and monofunctional E. coliaspartate kinase. Specifically, in two regions where particular aminoacid substitutions were known to yield lysine-insensitive monofunctionalE. coli aspartate kinase (see U.S. Pat. No. 5,773,691) themonofunctional corn aspartate kinase was found to have similar aminoacid sequence. These regions are shown below: Region 1 monofunctionalcorn aspartate kinase TSEVSVSVSLD monofunctional E. coli aspartatekinase TSEVSVALTLD

The lysine-insensitive mutant monofunctional E. coli aspartate kinasehas the underlined T (threonine) residue changed to I (isoleucine).Region 2 monofunctional corn aspartate kinase SSRMLGQYGFLAmonofunctional E. coli aspartate kinase SLNMLHSRGFLA

The lysine-insensitive mutant monofunctional E. coli aspartate kinasehas the underlined M (methionine) residue changed to I (isoleucine).

A site-specific mutation to change S (serine) to L (leucine) in the cornmonofunctional aspartate kinase at the position in Region 1 where a T(threonine) residue was changed to I (isoleucine) in monofunctional E.coli aspartate kinase was created using PCR mutagenesis as describedbelow.

First, a 370 bp portion of the corn monofunctional aspartate kinase cDNAin clone cpd1c.pk010.k1 was amplified via PCR using Oligo 2 (SEQ ID NO:19) and Oligo 3 (SEQ ID NO:22) as primers: (SEQ ID NO:22) Oligo 3:5′-TTAGTGTTTCTGTGTTACTTGATCCATCAAAG-3′

Then a 980 bp portion of the corn monofunctional aspartate kinase cDNAin clone cpd1c.pk010.k1 was amplified via PCR using Oligo 1 (SEQ IDNO:18) and Oligo 6 (SEQ ID NO:23) as primers: (SEQ ID NO:23) Oligo 6:5′-CTTTGATGGATCAAGTAACACAGAAACACTAAC-3′The 370 bp and 980 bp PCR fragments were then mixed together, denaturedand allowed to hybridize heterologously. Staggered ends were filled-inwith Taq polymerase, and PCR was performed on the DNA mixture usingOligos 1 (SEQ ID NO:18) and 2 (SEQ ID NO:19) as primers. This generateda 1320 bp Nco I-Kpn I fragment, designated PCR fragment 6, with thedesired mutation that changes S (serine) to L (leucine) in the cornmonofunctional aspartate kinase.

The 1320 bp NcoI-KpnI fragment containing the lysine-resistant (i.e.,reduced sensitivity to inhibition by lysine) mutant corn monofunctionalaspartate kinase was digested with Nco I and Kpn I and ligated intopTrcHis cut with the same enzymes. PCR fragment 3 described in Example 7was ligated into the resulting plasmid in the same way. PCR fragments 1and 3 were combined into a single plasmid described in Example 7. Thecreation of a mutant corn monofunctional aspartate kinase gene whichcontains a single nucleotide change compared to the native cornmonofunctional aspartate kinase gene resulting in a change of amino acid441 (in SEQ ID NO:8) from serine to leucine was confirmed by DNAsequencing. That the mutant corn monofunctional aspartate kinase geneencodes an enzyme with reduced sensitivity to inhibition by lysine wasconfirmed by in vivo testing as described below.

The mutant corn monofunctional aspartate kinase gene was inserted intothe pTrcHis vector, as was done for the wild type corn monofunctionalaspartate kinase gene, as described above. Plasmids carrying the mutantand wild type corn AK genes were transformed into Gif106M1 and testedfor their ability to support growth in the absence or presence ofexogenously added lysine. Both were able to support growth in theabsence of exogenously added lysine, indicating that both mutant andwild type enzymes were expressed and functional. However, only themutant corn monofunctional aspartate kinase gene was able to supportgrowth in the presence of exogenously added lysine, indicating that themutant enzyme was resistant to inhibition by lysine.

Example 9 Construction of Chimeric Aspartate Kinase Genes for Expressionin Plants

A chimeric gene for overexpression of monofunctional corn aspartatekinase in the embryo of transformed corn was constructed. The globulin 1promoter and 3′ sequences were isolated from a Clontech corn genomic DNAlibrary using oligonucleotide probes based on the published sequence ofthe globulin 1 gene [Kriz et al. (1989) Plant Physiol. 91:636]. Thecloned segment includes the promoter fragment extending 1078 nucleotidesupstream from the ATG translation start codon, the entire globulincoding sequence including introns and the 3′ sequence extending 803bases from the translational stop. To allow replacement of the globulin1 coding sequence with other coding sequences an Nco I site wasintroduced at the ATG start codon, and Kpn I and Xba I sites wereintroduced following the translational stop codon via PCR to createvector pCC50. An Nco I site within the globulin 1 promoter fragment wasthen eliminated by partial digestion with Nco I followed by singlestrand exonuclease treatment to remove the single-stranded overhangscreated by the Nco I digestion and then blunt end ligation creatingplasmid pHD1. The globulin 1 gene cassette is flanked by Hind III sites.

To construct the chimeric gene:

-   -   globulin 1 promoter/monofunctional corn aspartate        kinase/globulin 1 3′region

the 1320 base pair Nco I and Kpn I PCR fragment 1 (described in Example7) containing the major part of the monofunctional corn aspartate kinasecoding region was inserted into plasmid pHD1 between the globulin 1 5′and 3′ regions creating pBT954. A 380 bp fragment, designated PCRfragment 2, which has Nco I sites on both ends and contains the aminoend of the coding sequence including the plant chloroplast targetingsequence, was generated via PCR using oligo 7 (SEQ ID NO:24) and oligo 8(SEQ ID NO:21) as primers: oligo 7: 5′-GACTCCATGGCAATCCCAGTGCG-3′ (SEQID NO:24)PCR fragment 2 was digested with Nco I and ligated into pBT954.Insertion of 380 bp PCR fragment 2 in the proper orientation wasdetermined by DNA sequencing, yielding the plant expression vectorpBT960. Similarly, the 1320 base pair Nco I and Kpn I PCR fragment 6(described in Example 8) containing the major part of thelysine-resistant mutant corn monofunctional aspartate kinase wasinserted into plasmid pHD1 between the globulin 1 5′ and 3′ regionscreating pBT955. Then 380 bp PCR fragment 2 (above), which contains theamino end of the coding sequence including the plant chloroplasttargeting sequence, was digested with Nco I and ligated into pBT955.Insertion of 380 bp PCR fragment 2 in the proper orientation wasdetermined by DNA sequencing, yielding the plant expression vectorpBT961.

1. An isolated polynucleotide comprising: (a) a first nucleotidesequence encoding a first polypeptide comprising at least 50 aminoacids, wherein the amino acid sequence of the first polypeptide and theamino acid sequence of SEQ ID NO:10 have at least 95% identity based onthe Clustal alignment method, (b) a second nucleotide sequence encodinga second polypeptide comprising at least 95 amino acids, wherein theamino acid sequence of the second polypeptide and the amino acidsequence of SEQ ID NO:2 have at least 90% identity based on the Clustalalignment method, (c) a third nucleotide sequence encoding a thirdpolypeptide comprising at least 100 amino acids, wherein the amino acidsequence of the third polypeptide and the amino acid sequence of SEQ IDNO:4 have at least 70% identity based on the Clustal alignment method,(d) a fourth nucleotide sequence encoding a fourth polypeptidecomprising at least 100 amino acids, wherein the amino acid sequence ofthe fourth polypeptide and the amino acid sequence of SEQ ID NO:14 haveat least 80% identity based on the Clustal alignment method, (e) a fifthnucleotide sequence encoding a fifth polypeptide comprising at least 250amino acids, wherein the amino acid sequence of the fifth polypeptideand the amino acid sequence of SEQ ID NO:12 have at least 80% identitybased on the Clustal alignment method, (f) a sixth nucleotide sequenceencoding a sixth polypeptide comprising at least 400 amino acids,wherein the amino acid sequence of the sixth polypeptide and the aminoacid sequence of SEQ ID NO:6 or SEQ ID NO:8 have at least 85% identitybased on the Clustal alignment method, (g) a seventh nucleotide sequenceencoding a seventh polypeptide comprising at least 400 amino acids,wherein the amino acid sequence of the seventh polypeptide and the aminoacid sequence of SEQ ID NO:16 have at least 90% identity based on theClustal alignment method, or (h) the complement of the first, second,third, fourth, fifth, sixth, or seventh nucleotide sequence, wherein thecomplement and the first, second, third, fourth, fifth, sixth, orseventh nucleotide sequence contain the same number of nucleotides andare 100% complementary. 2-27. (canceled)
 28. A transgenic plant havingan altered level of at least one free amino acid in seed when comparedto a nontransgenic plant of the same species, said plant comprising anucleic acid fragment from aspartate kinase, said nucleic acid fragmentcapable of altering endogenous expression of said free amino acid andhas been introduced into the plant by transformation.
 29. A transgeniccorn plant having an increased level of free threonine in seed whencompared to a nontransgenic corn plant, said plant comprising a nucleicacid fragment encoding a polypeptide having aspartate kinase activity,wherein the polypeptide has an amino acid sequence of at least 80%sequence identity, based on the Clustal V method of alignment, whencompared to one of Seq Id No.: 6 or 8, and wherein said nucleic acidfragment is capable of altering endogenous expression of said free aminoacid and has been introduced into the corn plant by transformation. 30.The plant of claim 28 wherein said plant is a monocot or a dicot. 31.The plant of claim 28 wherein said plant is corn or soybean.
 32. Theplant of claim 28 wherein said free amino acid is threonine, aspartate,lysine or methionine.
 33. The plant of claim 28 wherein said free aminoacid is threonine.
 34. The plant of claim 28 wherein an altered level isan increased level or a decreased level of said free amino acid whencompared to the level of said free amino acid in a nontransgenic plantof the same species.